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《乳品生物化学》（英文版） 5 Salts of milk

The salts of milk are mainly the phosphates, citrates, chlorides, sulphates, carbonates and bicarbonates of sodium, potassium, calcium and mag- nesium. Approximately 20 other elements are found in milk in trace amounts, including copper, iron, silicon, zinc and iodine. Strictly speaking,

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5 Salts of milk 5.1 Introduction The salts of milk are mainly the phosphates, citrates, chlorides, sulphates, carbonates and bicarbonates of sodium, potassium, calcium and mag- nesium. Approximately 20 other elements are found in milk in trace amounts, including copper, iron, silicon, zinc and iodine. Strictly speaking, the proteins of milk should be included as part of the salt system since they carry positively and negatively charged groups and can form salts with counter-ions; however, they are not normally treated as such. There is no lactate in freshly drawn milk but it may be present in stored milk and in milk products. The major elements are of importance in nutrition, in the reparation, processing and storage of milk products due to their marked influence on the conformation and stability of milk proteins, especially caseins, and to a lesser extent the stability of lipids and the activity of some indigenous enzymes 5.2 Method of analysis he mineral content of foods is usually determined from the ash prepared by heating a sample at 500-600 C in a muffle furnace for about 4 h to oxidize organic matter. The ash does not represent the salts as present in he food because 1. the ash is a mixture, not of the original salts, but of the carbonates and oxides of the elements present in the food; 2. phosphorus and sulphur from proteins and lipids are t in the ash while organic ions, such as citrate, are lost during incineration; and 3. the temperature usually employed in ashing may vaporize certain volatile elements, e.g. sodium and potassium Therefore, it is difficult or impossible to relate the ash obtained from a food with its salts system, and low values are obtained for certain mineral elements by analysis of the ash compared to direct analysis of the intact food. Titrimetric, colorimetric, polarographic, flame photometric and atomic absorption spectrophotometric techniques are frequently used to analyse for the various mineral constituents; however, the quantitative estimation of

5 Salts of milk 5.1 Introduction The salts of milk are mainly the phosphates, citrates, chlorides, sulphates, carbonates and bicarbonates of sodium, potassium, calcium and magnesium. Approximately 20 other elements are found in milk in trace amounts, including copper, iron, silicon, zinc and iodine. Strictly speaking, the proteins of milk should be included as part of the salt system since they carry positively and negatively charged groups and can form salts with counter-ions; however, they are not normally treated as such. There is no lactate in freshly drawn milk but it may be present in stored milk and in milk products. The major elements are of importance in nutrition, in the preparation, processing and storage of milk products due to their marked influence on the conformation and stability of milk proteins, especially caseins, and to a lesser extent the stability of lipids and the activity of some indigenous enzymes. 5.2 Method of analysis The mineral content of foods is usually determined from the ash prepared by heating a sample at 500-600°C in a muffle furnace for about 4h to oxidize organic matter. The ash does not represent the salts as present in the food because: 1. the ash is a mixture, not of the original salts, but of the carbonates and 2. phosphorus and sulphur from proteins and lipids are present in the ash, 3. the temperature usually employed in ashing may vaporize certain volatile oxides of the elements present in the food; while organic ions, such as citrate, are lost during incineration; and elements, e.g. sodium and potassium. Therefore, it is difficult or impossible to relate the ash obtained from a food with its salts system, and low values are obtained for certain mineral elements by analysis of the ash compared to direct analysis of the intact food. Titrimetric, colorimetric, polarographic, flame photometric and atomic absorption spectrophotometric techniques are frequently used to analyse for the various mineral constituents; however, the quantitative estimation of

240 DAIRY CHEMISTRY AND BIOCHEMISTRY each ion in a mixture is frequently complicated by interfering ions. The major elements/ions in foods, including milk, may be determined by the following specific method Inorganic phosphate reacts with molybdate to form phosphomolybdate which may be reduced to a blue compound that can be quantified spectrophotometrically at 640 nm Calcium and magnesium may be determined by titration with EDTA or by atomic absorption spectroscopy on TCA filtrates or on wet-or dry-ashed sample Citrate forms a yellow complex with pyridine(which is carcinogenic)in the presence of acetic anhydride; the complex may be quantified spectro- photometrically. Alternatively, citrate can be determined by an enzymatic Ionized calcium may be determined spectrophotometrically after reaction with murexide or using a Ca2*-specific Sodium and potassium may be quantified by flame photometry, atomic absorption spectroscopy or ion specific electrodes e Chloride can be titrated with AgNO, using potentiometric or indicator end-point detection FeCl2, or by an enzymatic assay(using lactate dehydrogenase which can quantify both D-and L-isomers)or by HPLC References to these and other methods can be found in Jenness (1988) Detailed analytical procedures are published in the Official Methods of Analysis of the Association of Official Analytical Chemists(Arlington, VA SA)or in Standard Methods of the International Dairy Federation(Brus els, Belgium) 5.3 Composition of milk salts The ash content of milk remains relatively constant at 0. 7-0.8%, but the relative concentrations of the various ions can vary considerably. Table 5.1 shows the average concentration of the principal ions in milk, the usual range and the extreme values encountered. The latter undoubtedly include abnormal milks, e.g. colostrum, very late lactation milk or milk from cows with mastitic infection The ash content of human milk is only about 0. 2%; the concentration of all principal and several minor ions is higher in bovine than in human milk (Table 5.2). Consumption of unmodified bovine milk by human babies causes increased renal load and hence demineralized bovine milk or whey should be used for infant formulae

240 DAIRY CHEMISTRY AND BIOCHEMISTRY each ion in a mixture is frequently complicated by interfering ions. The major elements/ions in foods, including milk, may be determined by the following specific methods: 0 Inorganic phosphate reacts with molybdate to form phosphomolybdate which may be reduced to a blue compound that can be quantified spectrophotometrically at 640 nm. 0 Calcium and magnesium may be determined by titration with EDTA or by atomic absorption spectroscopy on TCA filtrates or on wet- or dry-ashed samples. 0 Citrate forms a yellow complex with pyridine (which is carcinogenic) in the presence of acetic anhydride; the complex may be quantified spectrophotometrically. Alternatively, citrate can be determined by an enzymatic assay. 0 Ionized calcium may be determined spectrophotometrically after reaction with murexide or using a Ca*'-specific electrode. Sodium and potassium may be quantified by flame photometry, atomic absorption spectroscopy or ion specific electrodes. 0 Chloride can be titrated with AgNO, using potentiometric or indicator end-point detection. 0 Sulphate is precipitated by BaCl, and quantified gravimetrically. 0 Lactate may be quantified spectrophotometrically after reaction with FeCl,, or by an enzymatic assay (using lactate dehydrogenase which can quantify both D- and L-isomers) or by HPLC. References to these and other methods can be found in Jenness (1988). Detailed analytical procedures are published in the Oflcial Methods of Analysis of the Association of Oficial Analytical Chemists (Arlington, VA, USA) or in Standard Methods of the International Dairy Federation (Brussels, Belgium). 5.3 Composition of milk salts The ash content of milk remains relatively constant at 0.7-0.8%, but the relative concentrations of the various ions can vary considerably. Table 5.1 shows the average concentration of the principal ions in milk, the usual range and the extreme values encountered. The latter undoubtedly include abnormal milks, e.g. colostrum, very late lactation milk or milk from cows with mastitic infection. The ash content of human milk is only about 0.2%; the concentration of all principal and several minor ions is higher in bovine than in human milk (Table 5.2). Consumption of unmodified bovine milk by human babies causes increased renal load and hence demineralized bovine milk or whey should be used for infant formulae

242 AIRY CHEMISTRY AND BIOCHEMISTRY 5.4 Secretion of milk salts The secretion of milk salts, which is not well understood has been reviewed and summarized by Holt (1985). Despite the importance of milk salts in determining the processing characteristics of milk, relatively little interest has been shown in the nutritional manipulation of milk salts composition. Three factors must be considered when discussing the milk salts system the need to maintain electrical neutrality, 2. the need to maintain milk isotonic with blood as a result of this, a set of correlations exist between the concentrations of lactose, Na, K- and Cl 3. the need to form casein micelles which puts constraints on the pH and [Ca2+] and requires the complexation of calcium phosphate with casein Skim milk can be considered as a two-phase system consisting of casein-colloidal calcium phosphate micelles in quasi-equilibrium with an aqueous solution of salts and proteins; the phase boundary is ill-defined because of the intimate association between the calcium phosphate and the caseins(phosphoproteins) Oo A fat-free primary secretion is formed within vesicles formed by blebbing- of the Golgi dicytosomes; the vesicles pass through the cytoplasm o the apical membrane where exocytosis occurs. The vesicles contain casein(synthesized in the rough endoplasmic reticulum toward the base of the mammocyte): fully-formed casein micelles have been demonstrated within the Golgi vesicles. The vesicles also contain lactose synthet (UDP: galactosyl transferase and a-lactalbumin)and there is good evidence showing that lactose synthesis occurs within the vesicles from glucose and UDP-galactose transported from the cytosol The intracellular concentrations of sodium and potassium are established by a Na/K-activated ATPase and Na and K can permeate across the vesicle membranes. Calcium is probably necessary to activate the UDP: galactosyl transferase and is transported by a Ca"/Mg-AtPase which concentrates Ca2+against an electrical potential gradient from uM concentrations in the cytosol to mM concentrations in the vesicles. Inor nic P(P) can be formed intravesicularly from UDP formed during the synthesis of lactose from UDP-galactose and glucose. UDP, which cannot cross the membrane, is hydrolysed to UMP and Pi, both of which can e-enter the cytosol (to avoid product inhibition) however, some of the P is complexed by Ca-t. Ca are also chelated by citrate to form largely oluble undissociated complexes and by casein to form large colloidal casein micelles Water movement across the vesicle membranes is controlled by osmotic pressure considerations. Since lactose is a major contributor to the osmotic pressure of milk, the concentrations of both soluble and colloidal salts in

242 DAIRY CHEMISTRY AND BIOCHEMISTRY 5.4 Secretion of milk salts The secretion of milk salts, which is not well understood, has been reviewed and summarized by Holt (1985). Despite the importance of milk salts in determining the processing characteristics of milk, relatively little interest has been shown in the nutritional manipulation of milk salts composition. Three factors must be considered when discussing the milk salts system: 1. the need to maintain electrical neutrality; 2. the need to maintain milk isotonic with blood; as a result of this, a set of correlations exist between the concentrations of lactose, Na', K+ and c1-; 3. the need to form casein micelles which puts constraints on the pH and [Ca"] and requires the complexation of calcium phosphate with casein. Skim milk can be considered as a two-phase system consisting of casein-colloidal calcium phosphate micelles in quasi-equilibrium with an aqueous solution of salts and proteins; the phase boundary is ill-defined because of the intimate association between the calcium phosphate and the caseins (phosphoproteins). A fat-free primary secretion is formed within vesicles formed by blebbingoff of the Golgi dicytosomes; the vesicles pass through the cytoplasm to the apical membrane where exocytosis occurs. The vesicles contain casein (synthesized in the rough endoplasmic reticulum toward the base of the mammocyte); fully-formed casein micelles have been demonstrated within the Golgi vesicles. The vesicles also contain lactose synthetase (UDP : galactosyl transferase and sr-lactalbumin) and there is good evidence showing that lactose synthesis occurs within the vesicles from glucose and UDP-galactose transported from the cytosol. The intracellular concentrations of sodium and potassium are established by a Na+/K+-activated ATPase and Na+ and K+ can permeate across the vesicle membranes. Calcium is probably necessary to activate the UDP : galactosyl transferase and is transported by a CaZ +/Mg2 +-ATPase which concentrates Ca2 + against an electrical potential gradient from pM concentrations in the cytosol to mM concentrations in the vesicles. Inorganic P (Pi) can be formed intravesicularly from UDP formed during the synthesis of lactose from UDP-galactose and glucose. UDP, which cannot cross the membrane, is hydrolysed to UMP and Pi, both of which can re-enter the cytosol (to avoid product inhibition); however, some of the Pi is complexed by Ca2+. Caz+ are also chelated by citrate to form largely soluble, undissociated complexes and by casein to form large colloidal casein micelles. Water movement across the vesicle membranes is controlled by osmotic pressure considerations. Since lactose is a major contributor to the osmotic pressure of milk, the concentrations of both soluble and colloidal salts in

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